The objective of the proposed research is to develop an integrated system for DNA sequencing by synthesis (SBS) using photocleavable fluorescent nucleotides. The SBS system includes the construction of a chip with immobilized single stranded DNA templates by site-specific coupling chemistry. These templates contain a self priming moiety to generate the complementary DNA strand in polymerase reaction using 4 photocleavable fluorescent nucleotides whose 3-prime-OH group is modulated to allow their efficient incorporation into the growing strand of DNA as temporary terminators in the polymerase reaction. A 4-color fluorescence imager is then used to identify the sequence of the incorporated nucleotide on each spot of the chip. Upon removal of the fluorophore photochemically and reactivation of the 3-prime-OH group, the polymerase reaction will proceed to incorporate the next nucleotide analogue and detect the next base. It is estimated that 10,000 bases will be identified after each cycle on one sequencing chip that contains 10,000 different DNA templates. The engineering part of the proposal will pursue the development of two generations of SBS prototypes. The first generation system, Gen 1, will utilize conventional thermal cyclers, slides, spotting systems and confocal scanners to assemble reactions and automatically read out the fluorescent signals as bases are extended along the DNA templates. This configuration will allow the optimization of the chemistry, fluidics and photolysis processes to generate data using the SBS method relatively quickly. In parallel with the Gen 1 chip-based prototype development, we will develop several of the key instrumentation technologies for the second generation system, Gen 2, including an ultra-high throughput, low volume PCR/spotting system and a CCD camera fluorescence measurement system. A Gen 2 prototype system will be designed and constructed with the capability of producing sequence data from about 500,000 samples in parallel. The success of this approach will allow the development of a high-throughput DNA sequencing system for genome sequencing and resequencing. [unreadable] [unreadable]